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| United States Patent |
5,605,812
|
|
Zomer
|
February 25, 1997
|
Test kit and method for the quantitative determination of coliform
bacteria and E. coli
Abstract
A test kit and method particularly for the quantitative determination of
total coliform bacteria and E. coli particularly in large volume test
samples, such as water, wherein the media includes an agent which changes
color and a gelling agent which reacts or provides in situ with the test
sample a semi-solid broth or media for incubation, thereby restricting the
mobility of the growth bacteria and permitting a quantitative count of the
separate, colored bacteria colonies.
| Inventors:
|
Zomer; Eliezer (Newton, MA)
|
| Assignee:
|
Charm Sciences, Inc. (Malden, MA)
|
| Appl. No.:
|
446039 |
| Filed:
|
May 19, 1995 |
| Current U.S. Class: |
435/38; 435/30; 435/34; 435/39; 435/810; 435/975 |
| Intern'l Class: |
C12Q 001/10 |
| Field of Search: |
435/29,34,38,39,7.37,30,810,975
|
References Cited
U.S. Patent Documents
| 3416998 | Dec., 1968 | Streitfeld.
| |
| 3959081 | May., 1976 | Witz et al.
| |
| 4072575 | Feb., 1978 | Lanham et al.
| |
| 4242447 | Dec., 1980 | Findl et al.
| |
| 4340671 | Jul., 1982 | Gibson.
| |
| 4643968 | Feb., 1987 | Weaver.
| |
| 4812393 | Mar., 1989 | Goswami et al.
| |
| 4812409 | Mar., 1989 | Babb et al.
| |
| 4923804 | May., 1990 | Leu et al.
| |
| 4925789 | May., 1990 | Edberg.
| |
| 5055414 | Oct., 1991 | Babb et al.
| |
| 5084041 | Jan., 1992 | Oxley et al. | 604/410.
|
| 5108902 | Apr., 1992 | Mooberry.
| |
| 5108903 | Apr., 1992 | Mooberry.
| |
| 5164301 | Nov., 1992 | Thompson et al.
| |
| 5210022 | May., 1993 | Roth et al.
| |
| 5223402 | Jun., 1993 | Abbas et al.
| |
| 5232838 | Aug., 1993 | Nelson et al.
| |
| 5284772 | Feb., 1994 | Oxley | 436/47.
|
| 5298392 | Mar., 1994 | Atlas et al.
| |
| 5354661 | Oct., 1994 | Doyle et al.
| |
| 5372801 | Dec., 1994 | Malmros et al.
| |
| 5393662 | Feb., 1995 | Roth et al. | 435/38.
|
| 5411867 | May., 1995 | Chang et al.
| |
| Foreign Patent Documents |
| WO9523026 | Aug., 1995 | WO.
| |
Other References
"Fluorocult.RTM. LMX-Broth Modified ace. to Manafi and OBner"--New Culture
Medium for the Simultaneous Detection of Coliform and E. Coli.
|
Primary Examiner: Wong; Leslie
Attorney, Agent or Firm: Crowley; Richard P.
Claims
What is claimed is:
1. In a test method for the quantitative determination of bacteria in a
sample, which test method comprises:
a) combining in a container a test sample, water where the test sample does
not comprise or constitute water, and a test composition, which
composition comprises a growth nutrient medium for the bacteria, and a
first chromogenic agent which is cleaved by enzymes in the bacteria to
produce and to indicate by the presence of a color from the cleavage of
the first agent, the presence in the test sample of bacteria; and
b) observing the color, or absence thereof, to determine the quantitative
presence of the bacteria in the test sample, the improvement which
comprises:
i) providing a sterile, flexible, transparent, sealable, plastic test
container which includes therein a powdered test composition and a gelling
agent in amount to provide in situ with the water or test sample and the
test composition a generally transparent gel medium;
ii) adding the test sample or water to said test container;
iii) sealing said test container;
iv) admixing the test sample or water with the powdered test composition
and gelling agent in said sealed container to form a generally transparent
gel medium;
v) forming the gel medium in said sealed container into a generally flat
gel medium layer;
vi) incubating the flat gel medium layer to provide for the growth, if any,
of bacteria in said test sample throughout the gel medium layer; and
vii) counting the color of the bacteria colonies in the incubated gel layer
to determine the quantitative amount of bacteria in the test sample.
2. The method of claim 1 wherein the gelling agent comprises a single
component, water soluble material.
3. The method of claim 1 wherein the gelling agent is a powdered gelling
agent and incorporated into said powdered test composition.
4. The method of claim 1 wherein the gelling agent comprises two or more
ingredients which ingredients react in situ in the test container to form
a gel-like media.
5. The method of claim 1 wherein the gelling agent is incorporated in the
test composition and comprises an admixture of a water soluble alginate
salt and a metal salt, which water soluble alginate and metal salt react
in the presence of water to form a metal alginate and to form the gel
medium.
6. The method of claim 5 wherein the gelling agent comprises a sodium or
potassium alginate, and the metal salt comprises a calcium salt.
7. The method of claim 5 wherein the gelling agent comprises an admixture
in the test composition of from about 2 gm/100 ml to 5 gm/100 ml of a
water soluble alginate, and about 0.5 gm/100 ml to about 1.0 gm/100 ml of
a metal salt to form a water insoluble metal salt alginate.
8. The method of claim 1 wherein the gelling agent comprises from about 0.1
g/100 ml to 10 g/100 ml of the medium.
9. The method of claim 1 wherein said plastic container comprises two
chambers separated by a sealing means, and which method includes providing
said two chamber container with said first sealed chamber containing said
test composition and gelling agent, and introducing the test sample into
said second chamber, and removing the sealing means to permit admixing of
the test sample, with the test composition and gelling agent in said first
chamber.
10. The method of claim 9 wherein said first chamber has a volume
sufficient to receive a test sample of from about 100 ml to 500 ml.
11. The method of claim 9 wherein the sealing means comprises a removable
clip divider or heat seal divider to form the two chambers, and which
method includes removing the clip or heat seal divider to permit admixing
of the powdered test composition, gelling agent, and water or test sample.
12. The method of claim 1 which includes forming the gel medium layer
without heating.
13. The method of claim 1 which includes sterilizing the sealed test
container after counting, and disposing of the sterilized sealed
container.
14. The method of claim 13 which includes sterilizing the sealed container
by boiling in water or autoclaving.
15. The method of claim 1 wherein the first agent comprises a
galactopyranoside and which test composition includes: an amplifying agent
to increase the activity of the enzyme synthesis; a second fluorogenic
agent which comprises glucuronide; and tryptophan.
16. The method of claim 9 which includes providing a thiosulfate in the
first chamber to neutralize oxidants in the test sample introduced into
the first chamber.
17. The method of claim 1 which includes selecting a gelling agent which
gelling agent provides for the formation of a gel-like medium in about two
hours or less after admixing with the test sample.
18. The method of claim 1 which comprises detecting the amount of coliform
bacteria or E. coli, or both, in the test sample.
19. The method of claim 18 which includes incubating the gel medium layer
with the use of an incubator at about 35.degree. C. to 45.degree. C. for
about 16 to 24 hours or without the use of an incubator at about
20.degree. C. to 30.degree. C. for 48 to 72 hours.
20. The method of claim 1 wherein the test sample comprises a water sample
having a volume of 10 ml to 500 ml.
21. The method of claim 18 wherein the first agent provides for a visible
blue-green color about the coliform bacteria.
22. The method of claim 18 wherein the first agent comprises a
halo-indolyl-.beta.-D-galactopyranoside.
23. The method of claim 18 wherein the test composition includes an
amplifying amount of a thiogalactopyranoside.
24. The method of claim 1 which includes a second agent which comprises an
alkyl-umbelliferyl-.beta.-D-glucuronide (MUG) and wherein the second agent
is cleaved by enzymes in E. coli bacteria and which second agent indicates
by the presence of a fluorescent color, the presence of E. coli bacteria
in the test sample, and which method includes measuring the fluorescent
color to determine quantitatively the amount of E. coli bacteria in the
test sample.
25. The method of claim 1 wherein the test composition includes tryptophan
to increase the sensitivity of the test method.
26. In a test method for the quantitative determination of total coliform
bacteria and E. coli, which test method comprises:
a) combining in a test container a water test sample to be tested for a
total coliform bacteria and E. coli with a powdered test composition,
which powdered test composition comprises a growth nutrient medium for the
coliform bacteria, and a first chromogenic agent which is cleaved by an
enzyme in the coliform bacteria to produce a color in the resulting broth
and to indicate by the presence of the color the presence in the test
sample of coliform bacteria, and a second fluorescent agent which is
cleaved by an enzyme in the E. coli bacteria to indicate by the presence
of a fluorescent color the presence of E. coli bacteria in the test
sample; and
b) observing the physical color, or absence thereof, and observing
fluorometrically the fluorescent color, or absence thereof, to determine
the qualitative presence of total coliform bacteria and E. coli bacteria
in the sample, the improvement which comprises:
i) providing a water soluble, powdered two component reactable gelling
agent in the test composition, which gelling agent reacts in situ in the
presence of the water test sample to form a transparent, semisolid gel
medium within about two hours of the combining of the water sample and
test composition and prior to incubation so as to restrict the mobility of
coliform bacteria and E. coli formed during incubation;
ii) providing a sterile, sealable, transparent, flexible plastic bag as the
test container, the plastic bag divided by a removable divider into a
first chamber and a second chamber, the second chamber containing the test
composition and the gelling agent;
iii) introducing the water test sample in an amount of from about 10 ml to
500 ml into the first chamber and sealing the first chamber;
iv) removing the divider;
v) admixing the test composition and gelling agent with the water test
sample to provide a gel medium containing the test sample;
vii) pressing the sealed plastic bag with the gel medium to form a
generally flat transparent gel medium layer in the sealed transparent
plastic bag;
viii) incubating the plastic bag with the gel medium layer to provide for
the growth of separate colonies of total coliform bacteria in visible
color throughout the gel medium layer and to provide a fluorescent zone
about colonies of the E. coli bacteria throughout the gel medium layer;
and
ix) quantitatively measuring the total coliform bacteria by counting the
colonies associated with the total coliform bacteria in the gel medium
layer, and quantitatively counting with long wave ultraviolet light, the
fluorescent zone about the E. coli fluorescent colonies.
27. The method of claim 26 which includes employing a gelling agent of a
metal salt alginate.
28. The method of claim 26 wherein the first chamber includes an agent to
oxidize halogens in the test sample.
29. The method of claim 26 wherein the first agent comprises a
halo-indolyl-.beta.-D-galactopyranoside, the second agent comprises an
alkyl-umbelliferyl-.beta.-D-glucuronide, and wherein the test composition
includes a thiogalactopyranoside amplifying agent and tryptophan.
30. The method of claim 26 which includes sterilizing the sealed plastic
bag after the measuring step and disposing of the sealed sterilized
plastic bag and the incubated gel medium therein.
31. A test kit for the qualitative determination of bacteria in a test
sample, which test kit comprises:
a) a sterile, sealable, flexible, transparent, plastic bag as a test
container and having an opening for the introduction of a test sample,
which test sample comprises water or has a water content, and means to
seal the opening in the plastic bag after introduction of the test sample;
b) a powdered test composition for admixture with the test sample in the
plastic bag, and which test composition comprises:
i) a growth nutrient medium for bacteria;
ii) a first agent which is cleaved by enzymes in the bacteria to produce
and to indicate by the presence of a visual color from the cleavage of the
first agent the presence in the test sample of bacteria; and
iii) a powdered gelling agent in the plastic bag which on admixture in the
plastic bag with the test sample and the test composition forms a
transparent, gel medium which may be formed into a flat, transparent gel
medium layer in the plastic bag prior to incubation to restrict the
mobility of the bacteria on growth in the flat gel medium layer on
incubation.
32. The test kit of claim 31 wherein the plastic bag includes removable
means to divide the bag into a separate test sample collecting chamber to
receive the test sample and a separate media chamber containing the
powdered test composition and gelling agent.
33. The test kit of claim 32 wherein the sample collecting chamber contains
an oxidant agent to neutralize oxidants in the water test sample.
34. The test kit of claim 31 wherein the means to divide comprises a seal
line between the said chambers.
35. The test kit of claim 31 wherein the means to divide comprises a
plastic clip divider.
36. The test kit of claim 31 wherein the means to seal comprises a wire
strip.
37. The test kit of claim 31 wherein the powdered test composition includes
as the gelling agent a water soluble alginate and a metal salt which form,
in the presence of the water test sample, a metal alginate.
38. The test kit of claim 31 wherein the powdered test composition includes
an agent for the detection of E. coli which is cleaved by enzymes in the
E. coli to indicate by a fluorescent color the presence of E. coli under
ultraviolet light in the test sample.
39. The test kit of claim 32 wherein the plastic bag is arranged and
constructed to receive a volume of from about 10 ml to 500 ml of the test
sample.
40. The test kit of claim 38 which includes:
a) a means to incubate the sealed plastic bag in a flat condition after
admixture of the test sample and the powdered test composition and gelling
agent; and
b) an ultraviolet light source means to detect E. coli in the test sample.
41. The test kit of claim 22 wherein the test composition comprises:
halo-indolyl-.beta.-D-galactopyranoside as a first agent and wherein the
second agent comprises an alkyl-umbelliferyl-.beta.-D-glucuronide, and the
test composition includes a thiogalactopyranoside amplifying agent and
tryptophan.
42. A method of preparing a gel medium layer for use in the determination
of bacteria, which method comprises:
a) providing an admixture in a water-containing test sample powdered test
composition which comprises: a growth nutrient medium for the bacteria and
a chromogenic agent which is cleaved by enzymes in the bacteria to
indicate, by the presence of a color from the cleavage of the first agent,
the presence in the test sample of bacteria; and a powdered gelling agent
admixed in an amount to provide in situ with the water-containing test
sample a generally transparent gel medium;
b) adding a water-containing test sample to the admixed powdered test
composition and gelling agent to form in situ a generally transparent gel
medium; and
c) forming the gel medium into a generally flat gel medium layer prior to
any incubation of the gel medium.
43. The method of claim 42 which includes carrying out the formation of the
gel medium in a container.
44. The method of claim 42 wherein the gelling agent comprises an alginate.
45. The method of claim 42 which includes forming the in situ gel medium
without heating and within a period of about two hours.
46. The method of claim 42 wherein the test composition is selected for the
determination of coliform bacteria.
47. The gel medium layer produced by the method of claim 42.
Description
BACKGROUND OF THE INVENTION
It is widely desirable to provide rapid, effective detection and
identification of various and numerous microorganisms in test samples, say
for example, from, but not limited to, water, food and body fluids, such
as for example, to detect and identify a total coliform bacteria and/or
also E. coli bacteria in a particular test sample.
One enzymatic test identification method is known as the MUG test which was
designed for the detection of E. coli. This test is well known and is set
forth in U.S. Pat. No. 5,223,402, hereby incorporated by reference. The
test method is for detecting total coliform bacteria and E. coli and other
microbes employing one or more chemiluminescent compounds in an enzymatic
test technique. The test is the detection of a qualitative presence or
absence of total coliform bacteria or E. coli or other microbes in a
sample by combining the sample with a 1-2-dioxetane compound which
decomposes to a light-emitting portion with the reaction of at least one
hydrolytic enzyme present in the microorganism in the sample, thereby
triggering light emission, so that the light emission can then be
detected. The test indicates the presence of the hydrolase activity of the
particular microbes in the sample on exposing the light-emitting sample to
a light-sensitive detector over a period of time.
An improved MUG test method is directed to the simultaneous detection of
total coliform bacteria and E. coli in a test sample, for example a water
sample, in a test method known as Fluorocult.RTM. LMX Broth (a culture
medium trademark of BDH Inc. of Brampton, Ontario, Canada). This improved
MUG test method is described for example in the paper "Simultaneous
Detection of Total Coliforms and E. coli--Fluorocult.RTM. LMX Broth" by
Dr. Rolf Ossmer et al presented at the 15th International Symposium/FOOD
MICRO 1993, The International Committee on Food Microbiology and Hygiene,
Aug. 31-Sep. 3, 1993 in Bingen/Rhine, Germany, hereby incorporated by
reference.
The improved MUG test provides for a selected enrichment broth which
permits the simultaneous detection in the qualitative manner of total
coliforms and E. coli in bacterial testing of water, food and other
materials. The broth has been formulated to provide a high nutritional
quality and phosphate buffers to guarantee a high growth rate for the
coliforms present and employs a lauryl sulfate to inhibit to a large
extent the growth of gram-positive bacteria. The simultaneous detection of
total coliforms and E. coli are made possible by the addition of
chromogenic substrates in the broth which permit the easier identification
of coliforms due to a color change from a yellow color to a blue-green
color about the coliforms. The use of the MUG compound provides more
specific identification of E. coli within the test sample. The broth
employs a halo-indolyl-.beta.-D-galactopyranoside (X-GAL) which is a
5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside which is cleaved by
coliforms producing a blue-green color in the broth after incubation. The
visual observation of this blue-green coloration would indicate the
presence of total coliform bacteria in the test sample, while the absence
thereof indicates the absence of total coliform bacteria to a lower limit.
The broth also employs an amplification agent, such as a
thiogalactopyranoside, such as a
1-isopropyl-.beta.-D-1-thiogalactopyranoside (IPTG) for an amplification
factor in the enzyme synthesis and increases the activity of the
.beta.-D-galactopyranoside base. The MUG agent which is a fluorogenic
substrate is an alkyl-umbelliferyl-.beta.-D-glucuronide, in particular, a
4-methylumbelliferyl-.beta.-D-glucuronide (MUG), which is cleaved by the
enzyme .beta.-D-glucuronidase, which is highly specific for E. coli.
The detection of E. coli is determined by measuring fluorometrically in the
long-wave UV range, which fluorescence indicates the presence of E. coli
and the absence of fluorescence indicates the absence of E. coli in the
test sample. The test broth employs a tryptophan concentration to improve
the indolyl reaction for additional confirmation of E. coli and increases
the sensitivity of detection in combination with the X-GAL and the MUG
reaction. In the prior art, a typical broth composition would then include
tryptose, sodium chloride, sorbitol for sugar fermentation, tryptophan,
di-potassium hydrogen phosphate and potassium dihydrogen phosphate, lauryl
sulfate sodium salt and X-GAL, MUG and IPTG. Thus, the Fluorocult.RTM.
test permits the determination of the presence or absence of total
coliform bacteria, Esherchia, Enterobacter, Klebsiella and Citrobacter as
well as E. coli bacteria.
The Fluorocult.RTM. LMX Broth in use provides a single strength preparation
which is a dehydrated culture medium which is then added to water and
subsequently poured into a test tube or test container and autoclaved for
15 minutes at 121.degree. C. Ideally, the LMX Broth should have a pH of
6.8.+-.0.1 at 25.degree. C., and the prepared broth is generally clear and
either colorless or slightly yellow. The test sample is added to the
prepared broth and incubation is carried out for 24 hours, and in some
cases 48 hours, at 35.degree. C. to 37.degree. C. The presence of
coliforms is determined by the broth turning blue-green due to the X-GAL
reaction while E. coli is detected by measuring the fluorescence which is
represented by a light blue fluorescence in the broth. The presence of E.
coli may also be confirmed by covering the culture with KOVACS indole
reagent, and the presence of E. coli detected by a cherry red color
appearing in the reagent, later after one or two minutes, to confirm the
presence of E. coli if desired.
None of the cited references are suitable to determine bacteria
quantitatively in a large volume of a sample, for example, about 10 ml to
500 ml as in water samples. In order to enumerate bacteria in large sample
volumes, e.g. over 10 ml, the sample needs to be filtered through a
0.22-0.45 .mu.m filter or to use multiwell plates.
The filtration technique is the standard technique being used by regulatory
agencies and microbiological analytical labs to numerate coliforms in 100
ml samples. Sample filtration with a 0.45 .mu.m filter is done first to
recover bacteria from 10 ml to 300 ml water or extraction solution (from
particulate food or soil sample) and then cultivate the filter (with the
bacteria) on an agar plate with growth media, such as the LMX
Fluorocult.RTM. or conventional media for coliforms, such as the M-endo
agar. This technique needs a sterile filtration assembly, a
microbiological hood to perform the filtration and preparation of agar
plates. It is laborious and needs a laboratory support, e.g. autoclave,
hood and vacuum system for filtration. Common problems encountered with
this filtration technique are from small particulates that block the
filter, such as silt, dust, rust or other suspended particulates.
Another method is using specific antibodies immobilized to plastic beads or
magnetic beads for specific recovery of target bacteria, followed by
cultivation of the beads in growth media or selective growth media. This
antibody method is expensive and again requires work under sterile
conditions and needs highly trained laboratory personnel.
A metabolic identification method is also common and used by
microbiological labs and commercial companies for identification of
thousands of microorganisms. This method requires enrichments and
purification of cultures (a single colony), and it is a laborious,
multistep procedure which can take 48 to 96 hours for identification of
individual bacteria. It has been reported by Biolog, Inc. of Hayward,
Calif. that over 1,100 species can be identified by metabolic tests using
specific enzymes and substrates utilization. However, the method is
expensive and requires highly trained and skilled laboratory personnel.
Another method known as the Colilert.TM. (a trademark of Idexx
Laboratories, Inc. of Westbrook, Me.) method uses substrate technology of
an Idexx Quanti-Tray for coliform enumeration in 100 ml water samples.
However, this method which is routinely used by the biotechnology industry
for isolation of bacterial or transformed cells is costly, and in the
specific case of the Colilert.TM. method requires a heat sealing system.
Numeration at the 25-100 cfu becomes a problem as multiple bacteria can
grow in a single cell, and a complex mathematical model is used to
correlate the visual results with the actual count (Most Probable Number
statistical model--MPN). The Quanti-Tray sealer required for this
operation can also become a source of cross contamination. This method is
described and claimed in WO95/23026, published Aug. 31, 1995.
It is desirable to provide a new and improved bacteria, test particularly
for coliform/E. coli that is simple to be performed by the layman, without
laboratory equipment and still gives qualitative as well as quantitative
results in a short period of time. For example, a test can be performed at
35.degree. C. to 45.degree. C. for 16-24 hours (incubator required) or at
a lower temperature of 20.degree. C. to 30.degree. C. for 48-72 hours.
It is desirable to provide an improved MUG test wherein the MUG test either
for total coliform bacteria alone or E. coli, or a combination thereof,
may be rapidly and effectively, not only qualitatively, but
quantitatively, determined.
SUMMARY OF THE INVENTION
The invention relates to a qualitative-quantitative test kit and method for
microorganisms, and in particular, concerns an improved MUG
qualitative-quantitative test kit and method for the determination of
total coliforms and/or E. coli particularly suitable in large sample
volumes, such as 10 ml to 500 ml water samples.
The invention concerns a test method for the quantitative determination of
coliform bacteria in a sample, which test method comprises combining in a
presterilized container a test sample, water where the test sample does
not comprise or constitute water, and a test composition. The test
composition comprises a sterile, dried or concentrate growth nutrient
medium for the bacteria, and includes a first agent which is cleaved by
enzymes in the coliform bacteria to produce and indicate by the presence
of a visual color change from the cleavage of the first agent, the
presence in the test sample of coliform bacteria. The method includes
observing the visual color, or absence in change thereof, to determine the
qualitative presence of the coliform bacteria in the test sample.
The improvement in the test method and system comprises adding to the test
container prior to incubation a gelling agent which in situ with the
water, test sample and the test composition provides a transparent,
gel-like or semi-solid broth or cultivation medium, so as to provide an
effective incubation environment which restricts bacteria mobility in the
environment, but provides nutrients and indicative substrates for the
growth of distinct colonies of bacteria and production of the identified
color metabolites throughout the gel-like broth or medium. The coliform
bacteria colonies form colonies of a visible color; and may be enumerated
or quantitatively counted by the visible color of the coliform bacteria
colonies to determine the quantitative amount of coliform bacteria in the
test sample.
In the LMX media, the reaction of the coloring agent produces a blue-green
color in the incubated test broth which indicates the presence of total
coliform bacteria, and a total light-blue fluorescent color for the
indication of the presence of E. coli . These are only qualitative tests.
To provide a quantitative assay with this media, the water needs to be
filtered and then incubate the filter with solid agar media in a plate. It
has been found that the determination of coliform bacteria and/or E. coli
, or preferably both, particularly with water samples, may be
quantitatively determined by employing a gelling agent in the broth,
either separately or incorporated into the test composition and admixed
with the test sample, which test sample is either a water sample or may
have water separately added. The presence of a gelling agent provides for,
after admixing and prior to incubation, the rapid increase in the
viscosity of the resulting broth to form a jelly-viscous, gelatin or
jelly-like, semi-solid broth or medium throughout incubation entrapping
individual bacteria and each multiplying as a separate, distinct colony in
the broth or medium. The total coliform bacteria and the E. coli which are
present in the test sample are separately grown throughout the depth of
the gel-like broth creating distinct colonies for the actual quantitative
counting of the coliform bacteria, which has a blue-green color, or an
actual counting of the E. coli with a fluorescent meter may be carried out
to arrive at both a qualitative and a quantitative determination of the
total coliform and E. coli in the test sample.
A wide variety of gelling agents may be employed in connection with the
invention to provide for the formation in situ of a generally transparent,
gel-like consistency in the incubated material in the container, which
gelling agent should not substantially or functionally affect the growth
of the bacteria during incubation or have any adverse affect on any of the
ingredients or the test results. The agent may be employed as a liquid or
a powder, alone or in various combinations, preferably a powder-type
material, either as a single or a multiple ingredient which reacts or
polymerizes upon contact with water to create a semi-solid matrix, so that
it may be added to a dehydrated culture medium, that is, to a test
composition with a color agent that changes from colorless or other
distinct color to a different color, and then is added directly to a water
or water-containing sample. The gelling agent should be transparent or
relatively colorless, so as not to affect any coloration in the incubated
broth, which would interfere with the quantitative counting of the total
coliform bacteria and/or E. coli in the test sample.
In one embodiment, the gelling agent may be a single component which is
added to provide a rapid thickening in the broth without any reaction, for
example, but not limited to:
a) a carboxymethyl cellulose, modified starch, polyvinyl alcohol and
derivatives and pectin;
b) 1% to 5% by weight of the medium of an alginate cross-linked or reacted
with a water insoluble metal ion, like calcium ions;
c) 1% to 5% carrageenans (biopolymers of D-galactose and anhydro galactose)
dissolved in buffered water containing sodium salt particles, such a
sodium carbonate, the sodium slowly dissolves and cross-links the
carrageenan to create a gel and entrap the bacteria; and
d) 1% to 5% chitozan (biopolymer of glucosamine, deacetylated chitin)
dissolved in buffered water with a pH of 6 and solidified by cross-linking
with metaphosphate or tripolyphosphate.
The gelling agent may be added for an in situ reaction or a cross-linking
carried out to provide for a gelation of the broth. The gelation time
typically should be such that the gelation occurs shortly after admixing
the test sample and prior to or during the early stages of the incubation,
such as for example, from zero to three hours after admixing, for example,
gelation starting thirty minutes to one hour after admixing of the
components making up the broth to be incubated with the test sample. The
amount of the gelling agent may vary as desired, sufficient added only to
provide for the desired gel-like or jelly consistency of the broth.
Without wishing to be bound by any particular concentration level, it has
been found for example that gelling agents in an amount as low as 0.1 gm
to for example up to 10.0 gm/100 ml of broth or more, would be suitable
say for example from 0.5 gm to 5.0 gm/100 ml.
Generally, the gelling agent forms the dispersed phase, in water and the
water in the test sample forms a continuous phase in the gel broth. Water
soluble gelling agents which would be suitable for the gelling agent and
employed in the practice of the invention may also include, but not be
limited to: collagen and collagen derivatives, such as hydrolysis products
of collagen; hydrocolloids derived from natural or synthetic materials,
such as alginates, which may be used alone, or alginic acid used alone in
an in situ reaction with various metal salts to form an in situ gel-like
consistency, such as for example, the reaction of alginic acid with a
metal salt, such as calcium, barium, magnesium or other salts to react
with a water soluble sodium or potassium alginates to form a gel-like
consistency in the broth.
The gelling agent may, for example, include polyvinyl-type compounds, such
as polyvinyl alcohol and its derivatives, and vinyl carboxylic-type
compounds, such as vinyl acetate, and other type compounds which are known
as thickening agents and gelling agents. The gelling agent should require
no preheating or dissolution of the gelling agent, as in the case of an
agar medium, and rapid gelling or polymerization at room temperature, e.g.
15.degree. C. to 45.degree. C., should occur. In the selection of suitable
gelling agents or combinations, polymers toxic to the bacteria or which
require organic solvents or which are hydrolyzed by the bacteria normally
should be avoided. Other suitable gelling agents may include pectins and
derivatives of pectin-type compounds which are suitable thickening or
gelling agents and which increase viscosity when admixed with
water-containing compositions and includes starches, gums, resins and
natural products, like carboxycellulose derivatives, such as carboxymethyl
cellulose, and would include silicates, and particularly colloidal
silicates which may form thickening and gel-type consistency in
water-containing materials.
The test samples employed may be derived from a wide of variety of sources
and include food, water, body fluids, such as urine, meat and milk. Where
the test sample does not include water, water may be separately added to
the dehydrated or powdered test composition when admixed with the test
sample to form the broth for incubation.
The invention will be described for the purposes of illustration only in
connection with the determination of total coliform bacteria and E. coli
bacteria in an improved LMX Fluorocult.RTM. media employing a water
sample. However, it is recognized that the test sample may be derived from
a wide variety of sources, and that water can may be separately added to
provide a gelling agent with the desired gel-like consistency to provide
for the separate formation of the coliform bacteria and E. coli bacteria
in the incubated broth.
The invention will be described for the purposes of illustration only in
connection with certain fluorogenic agents, that is, the MUG agent, as
well the coloring agent which is suitable for the detection of the total
coliform bacteria and to provide for a blue-green color. However, it is
recognized that now or in the future various or additional or new and
improved agents may be substituted for these agents for detection of total
coliform bacteria and E. coli bacteria, or other microorganisms in the
test sample that employ the essence of the present invention.
The test kit and method provide a rapid coliform specific test with the
results appearing overnight as a visible, distinct blue-green color for a
positive sample of total coliform and is designed to provide live
coliforms in 100 ml of water according to current Environmental Protection
Agency (EPA) requirements for drinking water. The agent may be a test
composition containing a dehydrated, powdered culture medium as
hereinafter described and containing the gelling agent therein sufficient
to provide a slow gel prior to or early in the incubation, typically
within the first hour or so of incubation. The equipment required is the
employment of a dehydrated test reagent and a long-wave UV light for the
detection of any fluorescence zone by the E. coli , as well as an
incubator operated at 37.degree. C., .+-.2.degree. C. No fluorescent
metering device is required, since the observation may be carried out
visually in a shaded area or in the dark using a portable, long-wave lamp.
It is recognized that there are new fluorgenic and chromogenic substrates
for glucoronidase which could provide identification of E. coli with
different color or change in color. For example, if E. coli is the main
target, using X-glucuronide will result in blue colonies for E. coli only
and green or blue fluorescent substrate for galactosidase can be used to
confirm the general coliforms (see example DetectaGene.TM. green or blue
from Molecular Probe, Inc. of Eugene, Oreg.). Also presently available is
ImaGene.TM. Red for red staining of bacteria with either galactosidase or
glucuronidase activities from Molecular Probe, Inc. supra. Thus, the media
identification substrate to be used can vary and be formulated according
to the priority target bacteria. Other bacteria similar to coliforms could
be identified by including other metabolic substrates in the media (for
example, the lactic acid bacteria.
In operation, the test kit and test method require a test container,
typically, for example, but not limited to a transparent, flexible,
throwaway, plastic, sealable bag which contains the dehydrated test
composition. The fingers are to be used to break up any possible clumps in
the dehydrated reagent in the bag, and the bag is labeled in connection
with the particular test. The bag is unsealed, and a defined amount, such
as for example, 100 ml of the water sample is then poured in to the bag.
As much air as possible is then removed from the bag, and the bag is then
closed tightly by folding tightly, for example with a wire strip or other
sealing means, to form a water tight seal. With the bag closed, the user
employs his fingers to knead or to break up any clumps larger than around
5 mm and to thoroughly mix the reagent medium by shaking the bag from side
to side, for example from 10 to 35 seconds, and to make sure that no dry
powder remains within the bag of the test container. The bag with the dry
medium and the water test sample with the bag sealed is then placed on its
side and incubated after being patted flat with the incubation at
37.degree. C., .+-.2.degree. C., for at least 16 hours or up to 24 hours.
The E*Colite.TM. (a trademark of Charm Sciences, Inc.) test kit of the
invention would include a dried, powdered test medium composition in the
bag comprising for example tryptose, sodium chloride, sorbitol,
tryptophan, di-potassium hydrogen phosphate, potassium dihydrogen
phosphate, lauryl sulfate sodium salt, X-GAL, MUG and IPTG, and a gelling
agent which comprises a water soluble alginate salt, such as sodium
alginate, and a calcium salt, like calcium carbonate, for use with a large
volume water sample to be tested.
The media bag can be divided into two separate chambers by a clip or
divider or heat seal line to form a sample collecting chamber and the
media chamber. This will have the advantage that the water sample can be
treated with thiosulfate to neutralize chlorine or bromine or other
oxidants in the sample that could interfere with the growth. The
thiosulfate is an important treatment recommended by the EPA. The sample
can be transported to the laboratory or incubation place where the divider
will be removed and the water sample in the collecting chamber will be
allowed to mix with the nutrients, indicator and gelling agent in the
media chamber. The use of a ColiGel.TM. (a trademark of Charm Sciences,
Inc. of Malden, Mass.) bag comes with both collection and incubation
chambers sterile and test ready and no transfer or filtration or sealing
procedures are required. Exposure of the sample to cross contamination is
eliminated. The only exposure of the sample will be at the site of
collection. Gelation occurs spontaneously upon mixing with water at room
temperature (10.degree. C. to 50.degree. C.), and no heating, such as in
agar preparation, is needed thereby avoiding the chance of excessive
heating which might kill target bacteria in the sample. The formation of
the gel also reduces accidental contamination as the bacteria are
entrapped in the gel phase which is easily contained. The bag can be
easily disposed of in regular trash after heat treatment, such as boiling
in water for ten minutes.
After incubation, if the interpretation and results is a negative
interpretation and results, that is, the absence of coliform bacteria,
would be indicated by no color change from yellow in a turbid medium with
no distinct blue-green color change seen. A positive result indicating the
qualitative presence of coliforms in the test sample would be separate and
distinctive blue-green color spots that formed in the gel-created media.
The number of blue-green spots show relative contamination levels per 100
ml of water, that is, a quantitative or semi-quantitative test for the
total coliform present. If after 16 hours incubation, the color is
inconclusive, that is, for example, weak blue-green color spots, the
incubation may be continued up to 24 hours or more and the incubator
results again examined. The detection of E. coli positive samples is
carried out by incubating the bag for an additional 16 hours, then
observing the bag under long-wave UV light (366 nm) for the indication of
a light blue fluorescent zone around the blue-green spots which indicates
the presence of E. coli and permits the detection of the semi-quantitative
amount of E. coli by employing a fluorescent meter against a standard
curve. Typically, following the test of all samples, positive and
negative, the sample broth should then be boiled in water for ten minutes
or autoclaved before disposal. As collection and incubation containers and
all media and indicators are sterile, no sterile conditions, like a
microbiological hood or an autoclave, are required.
The test kit and method will be described in particular reference to
certain illustrated examples; however, it is recognized that those people
skilled in the art may make various modifications, changes, improvements
and additions to the illustrated embodiments without departing from the
spirit and scope of the invention as described.
BRIEF DESCRIPTION OF THE DRAWING
The drawing is the schematic illustration of the test kit and method of the
invention.
DESCRIPTION OF THE EMBODIMENTS
The E*Colite.TM. test kit is a quantitative test of the simultaneous
determination of total coliforms and E. coli in water. It is equivalent in
performance to the standard membrane filter technique. The test uses
specific enzyme inducers in a semi-selective medium to detect the presence
of coliforms.
The test medium contains a slow polymerizing component which restricts
mobility of bacteria. Thus, each bacteria creates distinguishable colonies
for enumeration.
A coliform colony is positively identified based on the formation of a
visible and distinctive blue or green color, the product of
.beta.-galactosidase activity. E. coli colonies can be further confirmed
based on the formation of a zone of fluorescence around the blue/green
colonies, the product of .beta.-glucuronidase activity.
The test kit contains ready-to-use sterile medium packaged in a Whirl Pak
bag (also available in 2-compartment bags). The upper compartment is for
sample collection and treatment with thiosulfate to reduce the effect of
chlorine or bromine. The second compartment contains the nutrient and
enzyme substrates for the growth and identification of coliforms. The test
procedure does not require work under aseptic conditions, filtration or
any other sample preparation.
The method is applicable to drinking water, bottled water, ground water,
recreation water, surface water and other waters, for the purpose of
detection and enumeration of coliforms and/or E. coli at 1-300 cfu per 100
ml range. The test method and procedure are illustrated in the drawing
which is self-explanatory. A flexible, transparent, plastic, sealable bag
containing sterilized medium with polymerization agent is used. A sample
of 100 ml of water (or 300 ml in the 300 ml kit) is added to the bag. The
bag is sealed, and the medium is rehydrated with a short kneading and
mixing. The bag is laid flat and incubated for 16 to 24 hours at
37.degree. C. Bags are removed from the incubator and are ready for
observation and counting of blue/green coliform colonies. Fluorescence
around E. coli colonies may be identified with illumination with a UV lamp
(366 nm) after 24-48 hours of incubation.
The definitions of the test are:
Coliform and E. coli: negative--No blue/green spots appear in yellow gel
after 24 hours.
Coliform: positive--Blue/green spots appear in yellow gel after 16-24
hours.
E. coli: positive--Fluorescent blue/green colonies under UV light after
24-48 hours of incubation at 37.degree. C.
Toxic Material--Seven different industrial disinfectants were tested for
interference in the test method.
Soil Particulate--Various soils from light sand to heavy (clay) soil and
commercial compost were evaluated for interference in the test.
Suspensions were made by adding soil to water at 0.5 and 1 gram per 100 ml
and autoclaved for 10 minutes at 121.degree. C. After autoclaving, samples
were inoculated with E. coli (ATCC 11775) at 1-10 cfu/100 ml. All samples
(sterile control and inoculated) were incubated for 20 hours at 37.degree.
C.
Results indicate the soil had no interference in development, though
initial sample color was changed from yellow to brown. Because count is
determined by blue/green spots, the initial sample color is not
troublesome. All negative samples were negative with clear yellow color.
All inoculated samples were positive with blue/green spots. Similarly,
fluorescence was not affected by the presence of the soils.
Non-target Organisms--A blind study was conducted in house using sterile
bottled water to demonstrate the interference of non-target bacteria in
the test. Thirty sterile waters, thirty waters with 10 various non-target
strains and thirty waters contaminated with various coliforms at 1-20 cfu
level were randomly coded. The inoculum level was quantitated with 3M
coliform plates, and selected samples were also quantified by the membrane
filter technique. The results demonstrate no false positive in the thirty
negative samples, and no false positive in the thirty non-target samples.
This is better than 90% selection with 95% confidence and compares to
other standard methods. The membrane filtration technique had 0/10 false
positive in the negative samples, but had one false positive in the
non-coliform bacteria (presumably from carry over from earlier tested
samples on the same filter device).
Good laboratory practices should be observed although aseptic conditions
are not required. The polymerized medium greatly reduces the risk of
laboratory contamination or air contamination. However, after incubation
and recording the results, sample bags should be boiled for ten minutes or
autoclaved before disposal.
The instrumentation, equipment and supplies required are:
37(.+-.2).degree. C. incubator
A tray for samples is recommended.
A long wave length ultraviolet lamp (366 nm) is required for E. coli
confirmation.
The test kit contains:
a) semi-selective medium, enriched with tryptose and sorbitol as main
nutrient sources to support growth of coliforms and E. coli.
b) specific inducers and substrates for galactisidase
(5-Bromo-4-chloro-3-indolyl-.beta.- D-galactoside (X-GAL)) and
Glucuronidase (4-Methylumbelliferyl-.beta.-D-glucuronide (MUG))
c) biodegradable polymerizing agent in ready-to-use WHIRL-PAK.RTM. (a
registered trademark of Nasco Industries, Inc., Ft. Atkinson, Wis.) bag
d) sodium thiosulfate (10 mg) is also included in the medium for
neutralization of oxidizers such as chloride.
A standard positive enzyme is supplied separately and contains
galactosidase or glucuronidase to demonstrate proper blue/green color
development of coliforms and fluorescence development of E. coli.
Test kit reagents are stable for at least one year at room temperature.
Standard reference enzymes are stable for one year at 4.degree. C.
Sample Collection, Dechlorination, Preservation, Shipment and Storage
As indicated, sodium thiosulfate is optionally included in the medium. A
sample volume of 105.+-.5 ml water with air space is collected as directed
in standard methods or collected directly in the bag (double compartment
option) and transported to the lab for incubation as suggested. With the
double compartment bags, the sample is collected in the upper compartment
and treated with thiosulfate. Upon arrival to the lab, the plastic divider
is removed, and the water sample is reconstituted with the media in the
lower compartment. Samples collected in the upper compartment or other
containers should be refrigerated (2.degree. C. to 8.degree. C.) until
transferred to the testing location and reconstituted with the media.
Sample containers should be as specified in Standard Methods.
Test Method Performance Characteristics (Sensitivity, Specificity, Recovery
and Precision)
A preliminary study using the same growth medium composition, devoid only
of the gelling agent component, indicates detection of the following
bacteria with better than 95% confidence as positive for coliform:
Esherchia coli sp. (40 strains, all positive)
Enterobacter spp. (8 strains, all positive)
Citrobacter spp. (6 strains, all positive)
Klebsiella spp. (7 strains, all positive)
Other groups detected at less than 90%:
Serratia spp. (from 6 strains, 5 were positive)
Shigella spp. (from 3 strains, 2 were positive)
Hafnia spp. (from 2 strains, 1 was positive)
Forty seven other strains, all non-coliform, were tested negative.
A blind coded study was conducted to demonstrate the performance and
selectivity of the test method. The samples include:
30 samples containing sterile waters
30 samples inoculated with 10 non-coliform strains
30 samples inoculated with 3 E. coli spp., 3 Enterobacter spp., 2
Citrobacter spp. and 2 Serratia spp. (Tables 1-2)
For the coliform preparation, inoculum was targeting levels of 1-20 cfu/100
ml. Fresh cultures were standardized at 585 nm at 0.1 O.D., and then
diluted to 10-7. This was used as a stock inoculum for making the samples
in PBS, while for the non-coliforms, level 10-4 dilution was used as a
stock inoculum for making up the samples. One milliliter of each stock
inoculum was quantitatively estimated with Petrofilm.TM. (a trademark of
the 3M Company) 3M Coliform Count Plates. Sterile water samples of 100 ml
were inoculated with 1 ml of bacterial stock. Samples were tested by the
E*Colite test and the standard membrane filtration technique using M-endo
agar.
The results demonstrate no false positive in the thirty negative samples,
and no false positive in the thirty non-coliform samples (Table 1-2). This
is better than 90% selection with 95% confidence and compares to other
standard methods.
All coliforms were tested positive. From the two strains of Serratia (soil
coliform), one was tested positive (2 out of 3 samples) and one was
negative. The three E. coli strains had fluorescence and were positively
identified as E. coli . The results demonstrated good sensitivity for the
Charm C/E test. The membrane filtration technique gave low recovery of
bacteria in this study and was attributed to leakage in the filtration
assembly. An additional experiment was conducted to access the correlation
between E*Colite and the membrane filter technique.
An in-house study was designed to estimate the limit of detection of the
test kit with bacteria levels of 1-10 cfu/100 ml. Various strains of E.
coli and other coliforms were used with dilutions to bring the total
bacteria to approximately 1 cfu/ml or below. The inoculum was tested with
the Petrofilm.TM. 3M Coliform Count plates. The results clearly show
detection sensitivity at 1 cfu of target bacteria in 100 ml.
In another series of tests, E. coli (ATCC 121775) was used to evaluate the
quantitative aspect of the test kit, and the correlation with the membrane
filter technique. Concentration levels of 1 to 200 cfu/ml were used to
generate a correlation between the standard filtration technique and the
E*Colite test kit (Table 3). A Petrofilm.TM. 3M Coliform Count plate was
used for inoculum numeration. The regression coefficient of the test kit
versus the standard filtration technique was 0.969, and versus the
Petrofilm.TM. 3M, it was R=0.983. The regression analysis gave a y
constant close to 1, indicating the methods have similar detection limits
at about 1 cfu/ml.
The samples are totally contained in a solidified medium. The polymer used
as a gelling reagent is a carbohydrate base, food grade, and fully
biodegradable. Use of a plastic Whirl Pak bag eliminates any glassware
handling and makes it a pure, biodegradable test. Samples should be
autoclaved or boiled after use and discarded in regular trash.
The drawing illustrates and describes the test kit and method for the
qualitative determination of total coliform and E. coli in a water sample.
The test composition employed in dried, powdered form in each flexible bag
for use with 100 ml of a test water sample was:
______________________________________
Tryptose 0.5 g
Sodium chloride 0.5 g
Sorbitol 0.1 g
Tryptophan 0.1 g
Di-potassium hydrogen phosphate
0.27 g
Potassium dihydrogen phosphate
0.2 g
Lauryl sulfate sodium salt
0.01 g
X-GAL 0.008 g
MUG 0.005 g
IPTG 0.01 g
Gelling Agent
Sodium alginate 3.0 g
Calcium carbonate 0.8 g
Total per bag/100 ml water sample
6.503 g
______________________________________
Comparative, quantitative test data of the test results employed in the
test kit (E*Colite.TM. ) and method with the standard filter and Inoculum
by 3M with various bacteria are summarized in the following tables:
TABLE 1
__________________________________________________________________________
After 20 hrs at 37.degree. C.
Per 100 ml water
Inoculum by 3M E*Colite Standard Filter
Code
coliform cfu
Gas Formation
coliform
E. coli
total
coliform
No
Microorganism
ATCC
# (total cfu)
Around Colony
cfu Fluorescence
cfu cfu*
__________________________________________________________________________
1
E. coli 10536
28 13 + 12 Yes 3 3
2
E. coli 11775
58 10 + 23 Yes 9 9
3
E. coli 25922
100
11 + 4 Yes 3 3
4
Enterobacter
13045
69 16 + 22 No 35 1
5
Enterobacter
49701
30 15 + 8 No 2 0
6
Enterobacter
13047
53 22 + 10 No 3 0
7
Serratia
13880
44 8 No 0 No 17 0
8
Serratia
33105
64 9 No 2 No 8 0
9
Citrobacter
6750
73 20 + 12 No 7 7
10
Citrobacter
8090
60 15 + 2 No 25 25
11
B. cereus
11778
66 0 (700)
No 0 No 1 0
12
B. thuringiensis
35646
56 0 (400)
No 0 No TNTC
0
13
Ps. aeroginosa
27853
94 0 (>1000)
No 0 No 0 0
14
Ps. putida
17484
76 0 (250)
No 0 No 2 1
15
Enterococcus
29212
92 0 (800)
No 0 No
16
E. coli 10536
13 13 + 17 Yes
17
E. coli 11775
31 10 + 15 Yes
18
E. coli 25922
47 11 + 14 Yes
19
Enterobacter
13045
11 16 + 17 No
20
Enterobacter
49701
39 15 + 14 No
21
Enterobacter
13047
43 22 + 13 No
22
Serratia
13880
54 8 No 0 No
23
Serratia
33105
35 9 + 0 No
24
Citrobacter
6750
25 20 + 11 No
25
Citrobacter
8090
65 15 + 14 No
26
B. careus
11778
29 0 (700)
No 0 No
27
B. thuringiensis
35646
15 0 (400)
No 0 No
28
Ps. aeroginosa
27853
67 0 (>1000)
No 0 No
29
Ps. putida
17484
33 0 (250)
No 0 No
30
Enterococcus
29212
68 0 (800)
No 0 No
__________________________________________________________________________
*shainy colonies
NT not tested
NG No colonies observed
(Total cfu) 3M aerobic plate
TABLE 2
__________________________________________________________________________
Inoculum by 3M-cfu
E*Colite Standard Filter
code
coliform cfu
Gas Formation
coliform
E. coli
total
coliform
No
Microorganism
ATCC
# (aerobic)
Around Colony
cfu Fluorescence
cfu cfu*
__________________________________________________________________________
31
E. coli 10536
41 3 + 1 Yes
32
E. coli 11775
95 5 + 3 Yes
33
E. coli 25922
59 3 + 5 Yes
34
Enterobacter
13045
86 8 + 5 No
35
Enterobacter
49701
62 5 + 3 No
36
Enterobacter
13047
71 7 + 6 No
37
Serratia
13880
61 4 neg 0 No
38
Serratia
33105
63 2 neg 0 No
39
Citrobactor
6750
77 10 + 4 No
40
Citrobactor
8090
97 7 + 3 No
41
B. cereus
11778
1 0 (700)
NA 0 No
42
B. thuringiensis
35646
84 0 (400)
NA 0 No
43
Ps. aeriginosa
27853
38 0 (>1000)
NA 0 No
44
Ps. putida
17484
6 0 (250)
NA 0 No
45
Enterococcus
29212
55 0 (800)
NA 0 No
46
Baker yeast 21 0 (27) NA 0 No
47
B. catarhallis
25238
36 0 (195)
NA 0 No
48
Lactococcus
11603
82 NG NA 0 No
49
B. catarhallis
25238
27 0 (195)
NA 0 No
50
Baker yeast 22 0 (27) NA 0 No
51
St. aureus
12598
90 0 (40) NA 0 No
52
B. subtilis
6633
96 0 (96) NA 0 No
53
B. subtilis
6633
46 0 (96) NA 0 No
54
Lactococcus
11603
78 NG NA 0 No
55
St. aureus 32 0 (40) NA 0 No
56
Lactococcus
11603
88 NG NA 0 No
57
Baker yeast
7468
24 0 (27) NA 0 No
58
B. catarhallis
25238
19 0 (195)
NA 0 No
59
B. subtilis
6633
4 0 (96) NA 0 No
60
St. aureus
12598
23 0 (40) NA 0 No
__________________________________________________________________________
*shainy colonies
NT not tested
NG No colonies observed
(aerobic) 3M aerobic plate
TABLE 3
______________________________________
Numeration of E. coli by the ColiGel vs. Standard Filter method
An Example for E. coli (ATCC #11775)
LMX Charm
Inoculum
Fluoro-cult E*Colite/Coligel
Standard
3m Coliform E. coli Coliform
E. coli
Filter
Total blue Fluor. blue fluor Coliform
cfu neg/pos neg/pos cfu cfu cfu
______________________________________
0 neg neg 0 0 0
0 neg neg 0 0 0
0 neg neg 0 0 0
0 neg neg 0 0 0
0 neg neg 0 0 0
0 neg neg 0 0 0
8 pos pos 9 9 6
7 pos pos 11 11 12
8 pos pos 12 12 10
8 pos pos 13 13 10
8 pos pos 13 13 9
16 pos pos 17 17 23
16 pos pos 22 22 25
54 pos pos 44 44 48
54 pos pos 45 45 50
48 pos pos 51 51 61
105 pos pos 95 95 109
105 pos pos 96 96 100
126 pos pos 106 106 140
121 pos pos 114 114 137
105 pos pos 114 114 103
120 pos pos 124 124 129
128 pos pos 139 139 124
154 pos pos 141 141 177
168 pos pos 145 145 176
169 pos pos 154 154 185
163 pos pos 183 183 179
______________________________________
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